L0301P19 - Structure, Organisation and Replication of DNA
Central Dogma *DNA <———> RNA ———> Protein *Nucleus ———————> Cytoplasm Disruption of the Central Dogma *Retroviruses **RNA —> DNA before genetic information can be expressed **contains reverse transcriptase *Viruses **have RNA that get directly translated into protein and bypass DNA DNA *deoxyribonucleic acid *carbon, hydrogen, oxygen, nitrogen, phosphorus *transmits information between generations *packaged into chromosomes (DNA wrapped around histone protein) Structure Levels of Structure: #phosphate, ribose, and bases #phosphodiester linkages #base pairing to form the double helix #base sequences #chromosomes #genome Deoxyribose Group *5 carbon ring (1 - right on O, 5 - not in ring) *phosphate group attached to 5’ carbon *next nucleotide’s phosphate group attached to the 3’ carbon Nucleoside *adding a base to the deoxyribose sugar Nucleotide *adding a phosphate group to the nuceloside *dNTP - deoxy - nitrogenous base - triphosphate Nucleotide Chain *linked by phosphodiester bonds Nitrogenous Bases *include adenine, thymine, cytosine, guanine *T double bond A *C triple bond G *'Chargaff’s Rule:' the amount of purine = the amount of pyrimidines Double Helix *double stranded held together by hydrogen bonding *highly stable phosphodiester backbone *antiparallel Structure and Function *Complementarity **each strand provide information about the other *Complex sequences **variability of genetic information *Highly stable *Fidelity (loyalty) **the sequence of DNA is transmitted from cell to cell with minimal mistake **can detect who the parents are Eukaryotic DNA *human cell - total length of 2m *stored as chromosomes - segmented pieces of the genome bearing a linear sequence of genes in an X shaped formation *combined with histone proteins to form chromatin (compacted nucleosomes) - equal parts protein and DNA Nucleosomes *contains a core of 8 histones molecules (2 each of H2A, H2B, H3 and H4) with 146 bp of DNA wrapped around *Histone H1 clamps the DNA to the core *allows packaging of DNA into the nucleus RNA *ribonucleic acid *nearly always single stranded *ribose sugar instead of deoxyribose sugar *uses uracil instead of thymine *not all RNA made is transformed into protein Structure of Protein-Coding Genes *coding regions - exons *noncoding internal sequences - introns *noncoding flanking sequences that interact with machinery of transcription - promotors, operators, terminators *~65% of eukaryotic genes form families of related genes that have similar sequences and code for similar proteins *may be made at different times and in different tissues DNA Replication *semi conservative *each parent strand is a template for synthesis of a new strand *two replicated DNA helices contain one parent strand and one newly synthesised strand each Mechanism *prokaryotes - small circular chromosome with a single origin of replication *eukaryotes - large, linear DNA with many origins of replication where replication can occur at many different sites simultaneously Process #DNA helicase unwinds the double helix #single stranded binding proteins keep the template strands separated #RNA primase (a type of RNA polymerase) binds to template strand and synthesises a RNA primer 10-20 nucleotides long #once RNA primer is complete, primase is released #DNA polymerase¹ binds to RNA primers and adds nucleotides in the 5’ to 3’ direction #RNA primer is degraded and is replaced with DNA ¹DNA Polymerase *shaped like a hand where the finger region has precise shapes recognising the nucleotide bases *combined with a sliding DNA clamp (protein) stabilises the newly replicated strand Leading vs Lagging Strand Template right|600x800px|border *leading template is 3’ to 5’ **synthesis is 5’ to 3’ and is continuous *lagging template is 5’ to 3’ **synthesis is still 5’ to 3’ but is discontinuous **lagging strand is synthesised as Okazaki fragments **DNA polymerase I fills in the gap where RNA primers were **DNA ligase binds the Okazaki fragments Telomeres *end of the chromosome *repetitive sequences (eg TTAGGG) *shorten after each round of cell division by 50-200bps **at the end of the linear DNA molecule there is no place for a primer to bind thus new chromosomes have single strands of DNA at each end (after the primer is removed) **single stranded region is cut off along with some of the intact double stranded end *bind special proteins that maintain the stability of chromosome ends *approx. after 20-30 divisions, when it has shortened so much that they are no longer able to stabilise the ends, the cell dies Telomerase *catalyses the addition of any lost telomeric sequences *found in constantly dividing cells - bone marrow and gamete-producing cells *has a small piece of RNA (RNA template) Repair Mechanisms *lowers error rate to about one base 10^10 DNA Proofreading Mechanism *by DNA polymerase III *corrects errors during the replication process *about one error in 10^5 nucleotides *sense distortion and replaces incorrect nucleotide with the correct one Mismatch Repair Mechanism *scans and repairs errors in DNA shortly after replication *enzymes recognise distortion and cut out a small region (one strand) with the error *DNA polymerase I replaces the lost strand Excision Repair Mechanism *operates over the life of the cell to repair errors that result from chemical or radiation damage *larger distortion recognised by enzyme, cut out and replaced Practical Applications right|350x350px|border *principles of DNA replication can be used to design inhibitors of DNA replication, meaning cells cannot divide and will undergo programmed cell death *example: cisplatin **forms linkages between DNA strands and prevents replication **has a platinum atom bonded to two chlorines and two amino groups. **chlorines can be displaced easily by nitrogens from guanine bases to form strong covalent bonds **results in cross-linking the DNA strands, so that replication can’t occur **cross-linking cannot be repaired by the cell’s normal repair mechanisms **prevent tumour cells from growing DNA Sequencing *use of modified nucleosides (ddNTPs) **can be used by DNA polymerase and added to a growing DNA chain **lacks the hydroxyl group at 3’ position so no new nucleotides can be added after the ddNTP and synthesis stops Process #denature the DNA of interest #mix single stranded DNA with #*DNA polymerase #*primers #*large amount 4 normal dNTPs #*small amount of 4 ddNTPs (each tagged with a different fluorescent colour) #DNA polymerase synthesises strands of DNA mostly using dNTPs #ddNTP is incorporated, chain growth stops #newly made strands with the tagged ddNTPS separated from the collection of DNA strands #new strands sorted by length through gel electrophoresis #colour of fluorescent tags were read to determine the sequence of the bases High-Throughput Sequencing *developed for large genomes *uses miniaturisation techniques *fully automated, rapid and inexpensive *can sequence millions of bases in hours Advantages of Knowing the Genome *open reading frames **figure out coding regions of genes *amino acid sequences of proteins can be deduced by applying the genetic code *regulatory sequences: promoters and terminators for transcription *RNA genes *other non-coding sequences *mutations - changes in sequence between individuals